Identification of a Region Critically Involved in the Interaction of Phlorizin with the Rabbit Sodium-D-Glucose Cotransporter SGLT1

Abstract

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604-610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of alpha-methyl-D-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K(i) for phlorizin increased at least by a factor of 5 compared to the wild-type K(i) of 4.6 +/- 0.7 micromol/l; most striking changes were observed for Y604K (K(i) = 75.3 +/- 19.0 micromol/l) and C608K (K(i) = 83.6 +/- 13.9 micromol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (Km = 0.8 +/- 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside.

Three Surface Subdomains Form the Vestibule of the Na+/Glucose Cotransporter SGLT1

“…These cells have been shown in previous studies to lack endogenous Na ϩ -dependent glucose transport (6,16). All of the mutant clones showed lower AMG uptake rates, which might be due to insufficient sorting into the plasma membrane, inappropriate conformational structure of the expressed SGLT1 in the plasma membrane and/or a global reorganization of the disulfide bonds.…”

“…Transient transfection was performed by using the activated dendrimer transfection reagent SuperFect TM (Qiagen) as described earlier (16). The efficiency of transfection was tested in COS-7 cells that were simultaneously transfected with the pHook TM -2lacZ control plasmid expressing ␤-galactosidase under identical conditions (Invitrogen).…”

C-terminal Loop 13 of Na+/Glucose Cotransporter 1 Contains Both Stereospecific and Non-stereospecific Sugar Interaction Sites

“…For this purpose, end-tags with six histidines were added at both sides of the loop. The initial immobilization experiments done with a mixture of POPC and DOGS-Ni 2ϩ -NTA gave a strong indication for the expected interactions between His 6 -tagged loop 13 and Ni 2ϩ -NTA via the Ni 2ϩ -NTA/ His 6 system, as EDTA could prevent immobilization. Removal of the loop by competition with imidazole, which usually disrupts such interactions, failed, however; hence, additional interactions between the phospholipids and other parts of the peptide had to be assumed.…”

“…In previous studies we have shown that phlorizin binds to loop 13 of rabbit SGLT1 (aa 541-638) via the aromatic aglucone moiety inducing conformational changes of the loop, predominantly in the region between aa 600 and aa 621 (4,6) of the C terminus. Mutagenesis experiments provided also data on individual amino acids critically important for the interaction with the inhibitors (6,13,14).…”

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

“…Previous studies by our groups have shown that, in rbSGLT1, loops 13-14 facing the extracellular space is critically involved in the binding of the aglucon (64). In vitro studies suggest that the loop has a substructure of two small helices and interacts in several regions with membrane phospholipids (10,65).…”